For the positional cloning of genes and other novel types of genetic experiments, in humans and other organisms, there is a crucial need for techniques that can rapidly construct genome-wide high-resolution ordered clone maps. Current best methods, such as sequence-tagged site (STS) content mapping, entail a large number of experiments, and, in practice, require large low-resolution yeast artificial chromosome (YAC) clones and very many STSs. Here, Inner Product Mapping (IPM) is introduced that overcomes these limitations. IPM uses radiation hybrids (RHs) to provide localizing signatures for YACs. Two independent data tables are obtained which compare YACs against RHs, and RHs against STSs; these tables are combined to produce a computed map of the YACs against ordered STSs. IPM maps each YAC independently, requires relatively few RH comparisons to map a YAC, and can work with small (or large) YACs and few (or many) STSs.
There is currently considerable effort directed toward constructing genetic and physical genome maps for humans and model organisms. By localizing a phenotype on the genetic map, the corresponding physical and ordered clone maps can be used to retrieve chromosomal segments containing a gene of interest. DNA sequencing of these segments can determine the gene's DNA sequence, thus enabling functional studies of the gene's product and its mutations. A genetic map currently exists at 5 centiMorgan (cM) resolution (NIH/CEPH Collaborative Mapping Group (1992). A Comprehensive Genetic Linkage Map of the Human Genome. Science, 258: 67-86; Weissenbach, J., Gyapay, G., Dib, C., Vignal, A., Morissette, J., Millasseau, P., Vaysseix, G., and Lathrop, M. (1992). A second generation linkage map of the human genome. Nature, 359: 794-801), and a greater resolution map is currently being developed. Physical and ordered clone mapping of chromosomes, however, is still in its infancy (Maier, E., Hoheisel, J. D., McCarthy, L., Mott, R., Grigoriev, A. V., Monaco, A., Larin, Z., and Lehrach, H. (1992). Complete coverage of the Schizosaccharomyces pombe genome in yeast artificial chromosomes. Nature Genetics, 1: 273-277). Current technologies are enabling the construction of a 1 megabase (mb) resolution map (Bellanne-Chantelot, C., Lacroix, B., Ougen, P., Billault, A., Beaufils, S., Bertrand, S., Georges, S., Glibert, F., Gros, I., Lucotte, G., Susini, L., Codani, J.-J., Gesnouin, P., Pook, S., Vaysseix, G., Lu-Kuo, J., Ried, T., Ward, D., Chumakov, I., Le Paslier, D., Barillot, E., and Cohen, D. (1992). Mapping the Whole Genome by Fingerprinting Yeast Artificial Chromosome. Cell, 70: 1059-1068), but require tremendous experimental effort (Green, E. D., and Green, P. (1991). Sequence-tagged site (STS) content mapping of human chromosomes: theoretical considerations and early experiences. PCR Methods and Applications, 1: 77-90). For routine use, a higher resolution 250 kilobase (kb) map would be far more useful for locating genes. There is, therefore, a great and immediate need for ordered clone mapping strategies that are both more efficient and achieve higher resolution.
Crucial to the success of large scale mapping experiments is the availability of numerous sequence tagged sites (Olson, M., Hood, L., Cantor, C., and Botstein, D. (1989). A common language for physical mapping of the human genome. Science, 245: 1434-35) (STSs), which are unique, very short 100-500 base pair (bp) genomic sequences that can be amplified from sample DNA by the polymerase chain reaction (PCR). Polymorphic STS markers have proved highly successful in genetic linkage map construction (Weissenbach, J., Gyapay, G., Dib, C., Vignal, A., Morissette, J., Millasseau, P., Vaysseix, G., and Lathrop, M. (1992). A second generation linkage map of the human genome. Nature, 359: 794-801). Further, when also used as physical mapping reagents, these STSs can correlate the genetic and physical maps.
High resolution (&lt;250 kb) physical maps of STSs can be efficiently constructed via radiation hybrid (RH) mapping (Cox, D. R., Burmeister, M., Price, E. R., Kim, S., and Myers, R. M. (1990). Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science, 250: 245-250). A RH clone contains several very large (5 to 50 mb), nonoverlapping human chromosome fragments from a specific chromosome in a rodent cell line. These chromosome fragments are randomly formed, and cover 25-50% of the chromosome. To physically map STSs, a data table is constructed which compares a set of RH clones against a set of unordered STSs. Each entry in the table records whether or not an STS is present in some fragment of a RH. By permuting the order of the STSs (i.e., the table's columns) (Chakravarti, A., and Reefer, J. E. (1992). A Theory for Radiation Hybrid (Goss-Harris) Mapping: Application to Proximal 21q Markers. In Genetic Analysis Workshop 7: Issues in Gene Mapping and the Detection of Major Genes. Cytogenet Cell Genet, 99-101. vol. 59, (MacCluer, J. W., Chakravarti, A., Cox, D., Bishop, D. T., Bale, S. J., and Skolnick, M. H., eds.; Cox, D. R., Burmeister, M., Price, E. R., Kim, S., and Myers, R. M. (1990). Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science, 250: 245-250), an ordering can be found either by minimizing the number of obligate breaks (Boehnke, M. (1992). Radiation hybrid mapping by minimization of the number of obligate chromosome breaks. In Genetic Analysis Workshop 7: Issues in Gene Mapping and the Detection of Major Genes. Cytogenet Cell Genet, 96-98. vol. 59, (MacCluer, J. W., Chakravarti, A., Cox, D., Bishop, D. T., Bale, S. J., and Skolnick, M. H., eds.; Weeks, D. E., Lehner, T., and Ott, J. (1992). Preliminary ranking procedures for multilocus ordering based on radiation hybrid data. In Genetic Analysis Workshop 7: Issues in Gene Mapping and the Detection of Major Genes. Cytogenet Cell Genet, 125-127. vol. 59, (MacCluer, J. W., Chakravarti, A., Cox, D., Bishop, D. T., Bale, S. J., and Skolnick, M. H., eds.)) present in all RH rows, or by using maximum likelihood methods (Chakravarti et al., 1992; Cox, D. R., Burmeister, M., Price, E. R., Kim, S., and Myers, R. M. (1990). Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science, 250: 245-250). Inter-STS distances can also be estimated (Boehnke, M., Lange, K., and Cox, D. R. (1991). Statistical Methods for Multipoint Radiation Hybrid Mapping. Am. J. Hum. Genet., 49: 1174-1188; Chakravarti et al., 1992; Cox, D. R., Burmeister, M., price, E. R., Kim, S., and Myers, R. M. (1990). Radiation hybrid mapping: a somatic cell genetic method for constructing high-resolution maps of mammalian chromosomes. Science, 250: 245-250) by counting the RH breaks occurring between STSs.
Yeast artificial chromosome (YAC) clones (Bellanne-Chantelot et al., 1992; Burke, D. T., Carle, G. F., and Olson, M. V. (1987). Cloning of large exogenous DNA into yeast by means of artificial chromosomes. Science, 236: 806-812) can perpetuate a large (100 kb to 1.2 mb) linear sequence of human DNA. Since YAC DNA inserts are very large, an ordered YAC clone map is an ideal reagent for retrieving genes via positional cloning. Such YAC orderings can be constructed by labor and time intensive techniques such as hybridization fingerprinting, which compares restriction enzyme digests of two YACs at a time for commonalities in their fingerprint patterns, and extends contigs of overlapping YACs. The current best approach to ordering YACs is STS-content mapping (Green, E. D., and Green, P. (1991). Sequence-tagged site (STS) content mapping of human chromosomes: theoretical considerations and early experiences. PCR Methods and Applications, 1: 77-90), which employs YAC vs. STS comparisons. Two YACs overlap when they share a common STS; YACs containing two or more STSs can be used to extend contigs. When the STSs are genetically mapped, there is an immediate correspondence with the physical YAC clone map. The chief drawback to this method is that large YACs and very many STSs must be used to achieve sufficient intersections of YACs with STSs. Another likely candidate approach for mapping YACs at high resolution is RH mapping. Unfortunately, YAC inserts are too large to satisfy the necessary break minimization assumptions.
This invention utilizes Inner Product Mapping (IPM), a new technique that enables high resolution mapping of YACs using RHs. IPM employs a YAC vs. RH comparison data table, but supplements it with localizing information for each RH, specifying where its fragments reside on the chromosome: e.g., a RH vs. ordered STS table. When these two independent data tables are mathematically combined, a computed YAC vs. ordered STS table is obtained. The positive entries in this computed table localize the YAC along the chromosome or genome.
Lehrach's group has demonstrated the feasibility of performing highly parallelized experiments which can compare, for example, one RH against tens of thousands of gridded YACs in a singe experiment (Monaco, A. P., Lam, V. M. S., Zehetner, G., Lennon, G. G., Douglas, C., Nizetic, D., Goodfellow, P. N., and Lehrach, H. (1991). Mapping irradiation hybrids to cosmid and yeast artificial chromosome libraries by direct hybridization of Alu-PCR products. Nucleic Acids Res, 19(12): 3315-3318). This group has noted the utility of inner products, but has only used them for consistency checks, and not for building maps (Mott, R., Grigoriev, A., Maier, E., Hoheisel, J., and Lehrach, H. (1993). Algorithms and software tools for ordering clone libraries: application to the mapping of the genome of Schizosaccharomyces pombe. Nucleic Acids Research, 21(8): 1965-1974). Studies of the distribution of Alu and L-1 interspersed repetitive sequences (IRS) in YACs (Arveiler, B., and Porteous, D. J. (1992). Distribution of Alu and L1 repeats in human YAC recombinants. Mammalian Genome, 3: 661-668) and chromosomes have demonstrated the utility of using IRS-PCR as a mechanism for extracting human DNA content from various clonal reagents. Statistics for superimposing recombinant chromosomes in pedigrees have been used recently in genetic mapping (Nelson, S. F., McCusker, J. H., Sander, M. A., Kee, Y., Modrich, P., and Brown, P. O. (1993). Genomic mismatch scanning: a new approach to genetic linkage mapping. Nature Genetics, 4(May): 11-18; Ward, P. J. (1993). Some Developments on the Affected-Pedigree-Member Method of Linkage Analysis. Am. J. Hum. Genet., 52: 1200-1215), but, unlike IPM, make no use of neighborhood information and have not been applied to constructing physical maps.